This research focuses on oxidative modification of biopolymers, especially of proteins. The resulting covalent modifications have been implicated in important physiologic and pathologic processes. Determination of the actual roles of oxidative modification in these processes requires the identification of specific proteins which are susceptible to modification and the mapping of the sites of modification in those proteins. During this year, emphasis was placed on the development of novel methods for the detection and quantitation of those modification. In particular, we wished to quantify the fractional modification of each methionine residue in oxidatively modified proteins. While this might have been accomplished by reverse phase HPLC mapping of proteins cleaved by specific proteases, that is a challenging process complicated by varying recoveries and changing retention times caused by oxidation of the methionine residues. We therefore introduced the technique of simultaneous sequencing of a peptide collection. Simultaneous sequencing by an automated Edman sequencer generates quantitative results from which one can determine the location and extent of covalent modifications, provided that the sequence of the protein is known and the cleavage methods are selected to minimize or eliminate ambiguities. In addition to localizing methionine sulfoxide residues in glutamine synthetase and alpha-1-antitrypsin, the technique has been used to establish the histidine residues modified by metal-catalyzed oxidation of glutamine synthetase and the cysteine residue modified by glutathiolation in carbonic anhydrase. While the residues affected by metal-catalyzed oxidation are sited at metal-binding sites, those residues attacked by hydrogen peroxide or peroxynitrite are clearly surface exposed. Buried residues are resistant to such oxidation. Considerable effort was also directed towards development of a generalized method for localization of carbonyl groups within oxidatively modified proteins. Carbonyl groups are considered a hallmark for a variety of oxidations, so that localization of these modified residues is of general interest. Although the chemical reasons are not well understood, we found that reagents which effectively label low molecular weight carbonyl-containing compounds do not yield stable labeled derivatives of peptides. Fluorimetric and immunochemical methodologies were also pursued, but with limited success. However, radiochemical labeling was successful in identifying arginine-344 as the major carbonyl bearing residue in oxidatively modified glutamine synthetase. This method is being extended to localize modifications in other proteins under study, particularly carbonic anhydrase isozyme III and the iron-responsive proteins.